The superfamily of cupin proteins is the most functionally diverse of any family described to date and includes enzymes, transcription factors, seed storage proteins, auxin binding proteins, stress-related proteins (spherulins and germin-like proteins), and other proteins (Lapik R Y and Kaufman L S, Plant Cell. 2003 July; 15(7): 1578-1590.
The term cupin (from the Latin term “cupa,” for a small barrel or cask) has been given to a β-barrel structural domain identified in a superfamily of prokaryotic and eukaryotic proteins that include several enzymes, as well as factors that bind sugars and other compounds. This superfamily also includes many of the storage proteins from higher plants, and it was the knowledge of the three-dimensional structures of these proteins that allowed the molecular modeling of the wheat protein germin, an unusual protease-resistant protein with oxalate oxidase (OXO) (EC 1.2.3.4) activity. The main characteristic of the cupin domain is a two-motif sequence in which motif 1 corresponds to the C and D strands and motif 2 corresponds to the G and H strands of the unit structure of the bean storage protein phaseolin (Dunwell J M et al., Microbiology and Molecular Biology Reviews, March 2000, p. 153-179, Vol. 64, No. 1)
Although an expression in seed is suggested for several germin-like protein (GLP) proteins, there is no indication in the art neither for the specific expression profile nor for the strength of expression of the transcription regulating sequences disclosed herein. Furthermore, it is not guaranteed that the isolation of a promoter from its natural environment and its use in heterogeneous gene expression is workable.
Manipulation of plants to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in plant tissues. Such genetic manipulation relies on the availability of a means to drive and to control gene expression as required. For example, genetic manipulation relies on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant. For numerous applications in plant biotechnology a tissue-specific expression profile is advantageous, since beneficial effects of expression in one tissue may have disadvantages in others. Seed-preferential or seed-specific promoters are useful for expressing genes as well as for producing large quantities of protein, for expressing oils or proteins of interest, e.g., antibodies, genes for increasing the nutritional value of the seed and the like. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment. Moreover, the increasing interest in cotransforming plants with multiple plant transcription units (PTU) and the potential problems associated with using common regulatory sequences for these purposes merit having a variety of promoter sequences available.
There is, therefore, a great need in the art for the identification of novel sequences that can be used for expression of selected transgenes in economically important plants. It is thus an objective of the present invention to provide new and alternative expression cassettes for seed-preferential or seed-specific expression of transgenes in plants. The objective is solved by the present invention.